A family of variably expressed outer-membrane proteins (Vomp) mediates adhesion and autoaggregation in Bartonella quintana.

Abstract

Bartonella species are fastidious, Gram-negative human pathogens that can persist in the host bloodstream for years and bind to and invade several types of host cells. For many pathogens, adhesion to host cells and extracellular matrix (ECM) components is a critical virulence determinant. Bacteria often vary expression of surface adhesins by phase or antigenic variation to subvert the host immune response and permit adaptive interaction with different host structures. We developed a macaque animal model for Bartonella quintana infection to detect changes in bacterial outer-membrane proteins (OMP) during prolonged bloodstream infection. We identified a gene family encoding four highly conserved, 100-kDa, variably expressed OMP (Vomp), two of which function as adhesins. The variable expression of Vomp family members appears to be mediated by deletion of one or more vomp genes during chronic bloodstream infection. vomp deletion was observed also in isolates from humans with chronic B. quintana infection. The Vomp are closely related to the afimbrial adhesin, YadA, a virulence factor of Yersinia enterocolitica. The surface-expressed Vomp contain conserved structural features of YadA, including collagen-binding motifs. We demonstrate that the B. quintana Vomp are multifunctional OMP involved in binding to collagen and autoaggregation: VompC confers the ability to bind collagen IV, and VompA is necessary and sufficient for autoaggregation. The B. quintana Vomp are members of the newly recognized family of YadA-like trimeric autotransporters; the Vomp constitute a multigene family, they are variably expressed, and different virulence properties are attributable to individual Vomp family members.

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A family of variably expressed outer-membrane
proteins (Vomp) mediates adhesion and
autoaggregation in Bartonella quintana
Peng Zhang*, Bruno B. Chomel†, Maureen K. Schau*, Jeanna S. Goo*, Sara Droz*, Karen L. Kelminson*,
Smitha S. George*, Nicholas W. Lerche‡, and Jane E. Koehler*§
*Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, CA 94143-0654;†Department of Population Health and
Reproduction, School of Veterinary Medicine, and‡California National Primate Research Center, University of California, Davis, CA 95616
Communicated by Stanley Falkow, Stanford University, Stanford, CA, July 26, 2004 (received for review April 24, 2004)
Bartonellaspeciesarefastidious,Gram-negativehumanpathogens
that can persist in the host bloodstream for years and bind to and
invade several types of host cells. For many pathogens, adhesion
to host cells and extracellular matrix (ECM) components is a critical
virulence determinant. Bacteria often vary expression of surface
adhesins by phase or antigenic variation to subvert the host
immune response and permit adaptive interaction with different
host structures. We developed a macaque animal model for Bar-
tonella quintana infection to detect changes in bacterial outer-
membrane proteins (OMP) during prolonged bloodstream infec-
tion. We identified a gene family encoding four highly conserved,
100-kDa,variablyexpressedOMP(Vomp),twoofwhichfunctionas
adhesins. The variable expression of Vomp family members ap-
pears to be mediated by deletion of one or more vomp genes
duringchronicbloodstreaminfection.vompdeletionwasobserved
alsoinisolatesfromhumanswithchronicB.quintanainfection.The
Vomp are closely related to the afimbrial adhesin, YadA, a viru-
lencefactorofYersiniaenterocolitica.Thesurface-expressedVomp
contain conserved structural features of YadA, including collagen-
binding motifs. We demonstrate that the B. quintana Vomp are
multifunctional OMP involved in binding to collagen and autoag-
gregation: VompC confers the ability to bind collagen IV, and
VompA is necessary and sufficient for autoaggregation. The B.
quintana Vomp are members of the newly recognized family of
YadA-like trimeric autotransporters; the Vomp constitute a multi-
gene family, they are variably expressed, and different virulence
properties are attributable to individual Vomp family members.
phase variation ? multifunctional adhesin ? collagen binding ?
bloodstream infection ? YadA homolog
B
tonella pathogens for humans are Bartonella quintana, Bartonella
henselae, and Bartonella bacilliformis. B. quintana is transmitted
by the human body louse and causes relapsing fever (‘‘trench
fever’’), endocarditis, and bacillary angiomatosis (1). B. henselae
is transmitted by the cat flea or a cat scratch, causing cat-scratch
disease and bacillary angiomatosis (1). Prolonged infection can
occur, and unsuspected bloodstream infection with Bartonella
can be detected in 5–14% (2, 3) of individuals in certain regions.
In immunocompromised individuals (with cancer, transplanted
organs, or HIV infection), Bartonella infection can cause debil-
itating, even fatal, illness. Bartonellae must adapt to markedly
different environments, adhere to many cell types, and evade the
host immune response during persistent bloodstream infection
(even ?1 year). Because most Bartonella infections and species
were recognized only recently, little is known about Bartonella
virulence strategies.
The mechanism by which Bartonella species persist in the host
bloodstream is unknown, but successful evasion of the host
immune response is critical. Phase and antigenic variation are
among the most effective immune evasion strategies exploited by
artonella species are Gram-negative bacteria that are arthro-
pod-borne and extremely fastidious. The three major Bar-
microbial pathogens to maintain persistent host infection (4, 5).
Antigenic and phase variation involve a relatively high frequency
change in immunodominant surface proteins during infection
through alteration of the amino acid composition or by turning
on and off expression of a protein (4). Many bacteria, such as
Borrelia,Neisseria,andCampylobacter,usevariationmechanisms
(5–8). Virtually all of these antigenic- or phase-variable struc-
tures (e.g., adhesins) are virulence factors that are essential for
colonization or persistence of the pathogen in the host (8).
Variation of surface appendages also can direct tissue tropism
and facilitate adaptation to environmental changes, such as
those encountered in the transition between vector and host (5).
Another Bartonella persistence strategy is invasion of and resi-
dence in the intracellular compartment of nonphagocytic host
cells (9, 10). Binding of Bartonella to different host-cell types
likely involves adhesins that are variably expressed, but the
Bartonella adhesins that are involved have not been identified
definitively.
Neither antigenic nor phase variation have been documented
during Bartonella infection in vivo. We sought to identify outer-
membrane proteins (OMP) of B. quintana that are variably
expressed during prolonged bloodstream infection and that
interact with host cellular structures. By analyzing sequential
bloodstream isolates from an animal model, we identified and
characterized a family of B. quintana variably expressed OMP
(Vomp) adhesins that confer the two following important vir-
ulence phenotypes on B. quintana: autoaggregation and collagen
binding.
Materials and Methods
Bacterial Strains and Growth Conditions. B. quintana strains (Table
1, which is published as supporting information on the PNAS web
site) were streaked onto chocolate agar plates, incubated at 35°C in
candle jars, and harvested after 5–7 days (11). Escherichia coli
strains (Table 1) were grown in LB medium at 37°C. When
required, kanamycin, ampicillin, carbenicillin, or nalidixic acid was
added to growth medium at concentrations of 50, 100, 100, and 20
?g?ml,respectively.ToinduceexpressionofrecombinantVomp,E.
coli overnight cultures were diluted 1:100 with fresh LB medium
and the appropriate antibiotics. Isopropyl ?-D-thiogalactoside
(IPTG) was added for 1.5 h to a final concentration of 0.5–1 ?g?ml
when the OD at 600 nm reached 0.8.
Abbreviations: cfu, colony-forming units; OMP, outer-membrane protein(s); Vomp, vari-
ablyexpressedOMP;ECM,extracellularmatrix;MV;majorvariable;TOMP,totalOMP;IPTG,
isopropyl ?-D-thiogalactoside.
Datadeposition:ThesequencesreportedinthispaperhavebeendepositedintheGenBank
database (accession nos. AY618465 and AY618466).
§To whom correspondence should be addressed at: Division of Infectious Diseases, 521
Parnassus Avenue, Room C-443, University of California, San Francisco, CA 94143-0654.
E-mail: jkoehler@medicine.ucsf.edu.
© 2004 by The National Academy of Sciences of the USA
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Development of an Animal Model for Human B. quintana Infection.
WedevelopedananimalmodelofB.quintanainfectioninrhesus
macaques (Macaca mulatta) that reproduces the prolonged,
high-titer bacteremia that is observed in humans (unpublished
data). Animals were inoculated intradermally with inoculum
derived from multiple colonies of B. quintana strain JK-31
isolated from a patient and passaged three times to generate
sufficient inoculum. To study sequential B. quintana isolates
from experimentally infected animals, blood was removed from
animals twice weekly for 1 month after inoculation, then weekly
for 2 months. Blood was drawn into 2-ml EDTA tubes (Becton
Dickinson), centrifuged, and plated onto fresh chocolate agar.
The B. quintana colony-forming units (cfu) per ml of blood were
quantified, and at least five single colonies from sequential
isolates were selected for evaluation of OMP profiles. Preim-
mune and postimmune sera were drawn simultaneously with
cultures for the analysis of differentially expressed proteins.
Separation and Immunoblotting of B. quintana Cytosolic and Total
OMP (TOMP) Fractions. Fractionation of B. quintana proteins was
performed as described (12). The B. quintana TOMP fractions
were analyzed by 1D and 2D SDS?PAGE. For immunoblots, the
TOMP fractions were separated by 10% SDS?PAGE, and
proteinsweretransferredtoanitrocellulosemembrane,blocked,
and blotted with either a 1:50 dilution of serum from the
experimentally infected macaque or a 1:400 dilution of rabbit
antiserum (Animal Pharm Services, Healdsburg, CA) against
the N-terminal 15 aa of the Vomp (see Supporting Materials and
Methods, which is published as supporting information on the
PNAS web site).
Identification and Sequencing of the vomp Gene Family from JK-31
and B. quintana Final Isolate at Day 70 (BQ2-D70). Three Vomp
detected in the JK-31 TOMP preparation by using 2D SDS?
PAGE were transferred to a polyvinylidene difluoride mem-
brane, and the N-terminal sequence of each protein was deter-
mined (Kendrick Labs, Madison, WI). Additional internal-
peptide sequences were determined by digesting the Vomp with
endoproteinase Lys-C (Roche Molecular Biochemicals) and
analyzing the digests by matrix-assisted laser desorption ioniza-
tion MS. Several peaks representing internal-peptide fragments
present in all three Vomp were chosen for further sequencing.
The N-terminal and internal amino acid sequences were used to
synthesize degenerate oligonucleotide primers, 100kd5Deg and
100kd3Deg (Table 2, which is published as supporting informa-
tion on the PNAS web site), for library screening. The vomp
family gene sequences of JK-31 and BQ2-D70 were obtained by
screening a B. quintana JK-31 genomic library and by PCR
amplification (see Supporting Materials and Methods).
Southern Blotting with B. quintana Genomic DNA from Patient and
Sequential Animal Isolates. DNA was extracted from a population
of simultaneously isolated colonies (from the bloodstream of
animal BQ2) or from a single colony isolated after direct plating
of blood (from chronically infected patients). DNA was sub-
jected to Southern blotting (13) and probed with a PCR-
amplified, 645-bp fragment located within the conserved region
of the genes vompA, vompB, and vompC (primers: gene1consF
and gene1consR; see Table 2).
Surface Expression of the Vomp on Live JK-31, BQ2-D70, and Recom-
binant E. coli Strains. An indirect fluorescent Ab (IFA) technique
(14) was used to demonstrate Vomp surface expression on live
B. quintana and on vomp recombinant E. coli strains (for
construction of recombinant strains, see Supporting Materials
and Methods). B. quintana and IPTG-induced E. coli vomp
recombinant strains were incubated in anti-Vomp N-terminal
Ab (1:50) and then FITC-labeled secondary Ab (1:100)
(Zymed). After incubation, cells were washed, resuspended,
spotted onto a glass slide, air dried, and mounted. Bacteria were
visualized by using an Nikon Eclipse E600 epifluorescence
microscope (Nikon).
Autoaggregation Assay of B. quintana Strains JK-31 and BQ2-D70, and
Recombinant E. coli Strains Expressing Vomp. Autoaggregation was
assayed by a modification of the method described by Laird and
Cavanaugh (15). B. quintana were harvested, washed once, and
resuspended with M199 supplemented with 1 ?M glutamine and
1 ?M sodium pyruvate. We added 3 ml of each B. quintana
suspension (OD600? 1.0–1.2; ?108-109cells per ml) to a plastic
test tube and incubated them in a CO2-enriched atmosphere at
35°C. The IPTG-induced recombinant E. coli strains were
washed and resuspended in 3 ml of LB?carbenicillin supple-
mented with 68 ?g?ml chloramphenicol (to an OD600? 1.0–1.2;
?107-108cells per ml) and incubated at 37°C. To quantify the
autoaggregation, 50 ?l was taken from the top of each culture
tube from time 0 until 8 or 10 h, and OD600 was measured
immediately. Statistical analysis was performed as described (see
Supporting Materials and Methods).
Adhesion of B. quintana and Recombinant E. coli Strains Expressing
Vomp to Collagen IV. Bacterial adherence to collagen IV (human
placenta; BD Biosciences) was tested by a modification of the
method of Eberhard et al. (16). Glass coverslips in 24-well plates
were coated overnight at 4°C with 300 ?l of 20 ?g?ml collagen
IV (in coating buffer of 0.25 M NaHCO3?0.25 M Na2CO3, pH
9.6). Coverslips were washed with PBS containing 0.05% Tween
20 (PBST), blocked with 1% BSA for 2 h at room temperature,
and then washed three times in PBS. Bartonella and IPTG-
induced E. coli vomp recombinant strains were harvested,
washed with PBS, and resuspended in PBS to OD600 ?1.5
(?107-109cells per ml). For E. coli strains, mannose was added
to a final concentration of 1% to block background binding (17).
We added 1 ml of bacterial suspension to each well, and the
plates were incubated at room temperature for 2 h with gentle
shaking. After washing with PBST, bound bacteria were stained
with a 1:500 dilution of Syto-9 (Molecular Probes) for 15 min.
Coverslips were washed and mounted, and the binding of
bacteria was observed by epifluorescence microscopy. Quanti-
fication and statistical analysis of the bound cells (Supporting
Materials and Methods) were performed by counting three
random fields of each slide for three independent experiments
under ?400 and ?1,000 magnification for E. coli and B. quin-
tana, respectively.
Results
An Animal Model to Identify Changes in B. quintana Proteins During
Prolonged Bloodstream Infection. We established a rhesus ma-
caque animal model that reproduces the manifestations of B.
quintana infections in humans (unpublished data). Fig. 1A shows
the B. quintana cfu?ml values for blood isolated from a repre-
sentative infected rhesus macaque (BQ2), drawn after experi-
mentalinoculationwith6.6?107cfu?ml.InoculumstrainJK-31,
day 12 isolate (BQ2-D12), and day 70 isolate (BQ2-D70) were
used for further analyses in this study. The inoculum consisted
of multiple colonies that were isolated directly from a patient
andinwhichtheBQ2-D70genotypewasnotdetectable.Analysis
of isolates from another animal that simultaneously received the
same inoculum did not identify a BQ2-D70 genotype (data not
shown).
Immunoblottingand2DSDS?PAGEIdentifiedaFamilyof100-kDaOMP
That Disappear During Infection. To screen for differences in OMP
expression during prolonged bacteremia in the macaque, whole-
cell lysates from sequentially isolated strains were fractionated
into TOMP and cytosolic proteins. Direct comparison of JK-31
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and BQ2-D70 TOMP by 1D SDS?PAGE did not reveal obvious
differences (data not shown). Next, we immunoblotted the
sequential isolate TOMP with preimmune serum and day 70
postimmune serum from the animal (Fig. 1B). With postimmune
serum, a protein of ?100 kDa was detected in JK-31 TOMP but
not in BQ2-D70 (Fig. 1B). Abs to the 100-kDa protein were
elicited in all other experimentally infected animals (data not
shown), implying that this protein is an immunodominant anti-
gen. Analysis by 2D SDS?PAGE revealed an OMP of ?100 kDa
that appeared to be present in three isoforms in JK-31, each
migrating with a slightly different pI and apparent molecular
mass (Fig. 2A). However, for BQ2-D70, none of these isoforms
was visible (Fig. 2B).
The Vomp Are Encoded by a Family of Four Highly Conserved,
Tandemly Arranged Genes and Have Features Suggesting Surface
Localization. We identified four tandemly arranged, closely related
vomp genes, vompD, vompA, vompB, and vompC (named in order
of identification), located on an ?12.8-kb region of JK-31 genomic
DNA (Fig. 3A). There is a 43-bp intergenic region between vompA
and vompB and three putative promoter regions (Fig. 3A; PD, PAB,
and PC). There also is a 267-bp vomp gene fragment located 914-bp
downstreamofvompC.ThepredictedmolecularmassoftheVomp
ranges from 95,943 to 100,516 Da (see Table 3, which is published
as supporting information on the PNAS web site), with an acidic pI
(4.77–5.08).Overall,thereis47–90%aminoacididentityamongthe
four Vomp (see Table 4, which is published as supporting infor-
mation on the PNAS web site); VompA, VompB, and VompC are
more closely related to each other (84–90% amino acid identity).
The C-terminal VompD segment of ?350 aa has 87–89% identity
with the C-terminal regions of the other Vomp, but the N-terminal
448 aa of VompD has no homology to VompA, VompB, and
VompC.EachVomphasmajorvariable(MV),minorvariable,and
extensively conserved regions (Fig. 3A). In VompA, VompB, and
VompC, there also are two tandemly repeated regions at the C
terminus,inanX-Y—X-Ypattern(Xiscomposedof33–45aa,and
Y is composed of 61 aa; Table 3). Each B. quintana Vomp is
predicted to have a cleavable leader sequence (SIGNAL P program;
ref. 18), which was confirmed for VompA, VompB, and VompC by
N-terminal sequencing of the mature proteins. Interestingly, no
Vomp has an Arg–Gly–Asp (RGD) motif that might mediate
host-cell binding (19).
Vomp Family Members Have Conserved Motifs and Domains Present
in OMP Adhesins of Other Gram-Negative Pathogens. VompA,
VompB, and VompC were found to be most closely related to a
group of newly recognized afimbrial adhesins of Gram-negative
bacteria, including the well characterized YadA from Yersinia
enterocolitica (20), UspA from Moraxella catarrhalis (21), and
NadA from Neisseria meningitidis (22). There also is homology
with putative adhesins from E. coli O157:H7, Xylella fastidiosa,
and Salmonella typhimurium. YadA, a multifunctional OMP,
confers a number of critical virulence properties, including
adherence to epithelial cells (23) and professional phagocytes
(24), serum resistance (25), autoaggregation (26), and binding to
extracellular matrix (ECM) proteins, including collagen (27). Of
note, YadA is about half the size of Vomp, but the YadA N- and
C-terminal halves share homology with the corresponding re-
gions of VompA, VompB, and VompC (Fig. 7, which is pub-
lished as supporting information on the PNAS web site).
The predicted secondary structure of Vomp revealed that the
lollipop-shaped surface structure of YadA is likely also present
in VompA, VompB, and VompC (Fig. 7). Like YadA, the
C-terminal region of all four Vomp is predicted to consist of a
membrane anchor of four transmembrane ?-strands and an
?-helical internal region with a propensity to form coiled-coils
(20). Most importantly, four repeats of the NSVAIG-S collagen-
binding motifs in YadA (28, 29) are found within the N-terminal
half in VompA, VompB, and VompC (Figs. 3A and 7), followed
by a conserved neck and stalk region. No NSVAIG-S collagen-
binding motifs were identified in VompD.
Genomic Rearrangement of the vomp Locus, with Deletion of vompA
and vompB, Occurs During Chronic Bloodstream Infection. Southern
blot analysis (see Fig. 8, which is published as supporting informa-
tion on the PNAS web site) and sequencing of the vomp locus in
BQ2-D70revealedthatthelocusisonly6.3kbandthatonlyvompD
and vompC are present, with their putative promoters intact (Fig.
3B).AbsenceofvompAandvompBinBQ2-D70indicatesthatgene
rearrangement and?or deletion occurred in vivo during the course
of B. quintana bloodstream infection. We found that progressive
genomic rearrangement occurred during infection (see Fig. 8).
Hybridization of probe to multiple sized fragments of genomic
DNAfromapopulationofisolatesfromday12islikelytorepresent
a heterogeneous population of B. quintana in which some blood-
streamisolateshavenogenesdeleted,somehaveonegenedeleted,
and some have two genes deleted. By day 70 after inoculation, the
population of bloodstream isolates has become more homoge-
neous,andtheprobepredominantlyhybridizestoasinglefragment.
vomp Gene Deletion and Heterogeneity in the vomp Locus Occur in
Patient B. quintana Isolates. To identify whether heterogeneity
occurs within the vomp locus of B. quintana isolated from the
bloodstream of chronically infected patients, Southern blotting
was performed with genomic DNA from B. quintana human
isolates. Six different patterns of probe binding were identified
(Fig. 4), presumably resulting from differences among the pa-
Fig. 1.
infectionleadstoidentificationofanOMPpresentintheinoculumstrainthat
is not detectable in a late isolate by immunoblotting. (A) A representative
animal (BQ2) was inoculated with B. quintana JK-31 at time 0, and blood
cultures were performed at regular intervals for 98 days. cfu of B. quintana
bacteria per ml of blood are shown on the y axis. Bloodstream infection was
detectedonday12afterexperimentalinoculation,andarelapsingpatternof
bacteremia was observed, with the last isolate recovered on day 70. (B)
Immunoblot analysis demonstrates that a 100-kDa protein (arrow in II) is
present in JK-31 but absent in BQ2-D70. TOMP from JK-31 (lanes 2 and 4) and
BQ2-D70 (lanes 3 and 5) were immunoblotted with preimmune serum (Panel
I); or Day 70 postimmune serum (Panel II). Lane 1 shows protein standards.
Development of a rhesus macaque model of prolonged bloodstream
Fig. 2.
absent in BQ2-D70. TOMP fractions were subjected to 2D SDS?PAGE and
visualized by silver staining. (A) TOMP profile from JK-31. (B) TOMP profile
from BQ2-D70. The bracket and arrow indicate three 100-kDa proteins visible
in JK-31 but not BQ2-D70.
A family of 100-kDa proteins identified in JK-31 by 2D SDS?PAGE are
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tient isolates in the number of vomp genes; in the JK-5, JK-7, and
JK-56 isolates, a gene in the vomp locus appears to be deleted.
In other human isolates, all vomp genes are likely to be present,
but the regions flanking the vomp locus apparently differ in size.
Restriction-fragment polymorphisms among strains may also be
involved in pattern changes.
Vomp Mediates B. quintana Autoaggregation, and VompA, not
VompC, Is the Major Determinant of the Autoaggregation Phenotype.
We demonstrated by IFA that Vomp is expressed on the surface
of B. quintana JK-31 and vomp recombinant E. coli strains but
not on BQ2-D70 (see Fig. 9, which is published as supporting
information on the PNAS web site). We next assayed for an
autoagglutination phenotype, and we observed that JK-31 began
to sediment within the first hour, resulting in a significant
decrease in OD relative to BQ2-D70, which remained suspended
(Fig. 5B; P ? 0.05). After 36 h of incubation, JK-31 formed a
flocculate pellet at the bottom of the tube, but BQ2-D70
remained in suspension (Fig. 5A). This result suggests that the
VompA and?or VompB expressed in JK-31 but not in BQ2-D70
mediates the autoaggregation phenotype.
We then assayed for ability of Vomp expression to confer
gain-of-function on nonaggregative E. coli. Quantitative analysis
by fluorescence-activated cell-sorting demonstrated that surface
expression of VompA and VompC are equivalent on the in-
duced, recombinant E. coli strains (data not shown). Sedimen-
tation profiles in Fig. 5C show that the sedimentation rate of the
vompA, but not vompC, recombinant was significantly greater
than the E. coli control (P ? 0.05). This assay demonstrates that,
of VompA and VompC, VompA is the major determinant of the
autoaggregation phenotype of B. quintana. This result is consis-
tent with the finding that JK-31 (vompA?), but not BQ2-D70
(vompA?), autoagglutinates.
VompC and VompA are Collagen-Binding Adhesins with Different
Binding Efficiencies. Because the YadA NSVAIG-S collagen-
binding motifs are present in B. quintana Vomp (Fig. 3), we tested
JK-31 and BQ2-D70 for the ability to bind to ECM components,
andwefoundthatbothbindtocollagenIV,abasement-membrane
component(datanotshown).BecauseVompisnotdetectedonthe
surfaceofBQ2-D70,bindingcouldbeduetootheradhesinspresent
on B. quintana that confer redundant collagen-binding ability
(redundant adhesins are present in many bacteria) (30). To char-
acterize the contribution of individual Vomp, we tested whether
expression of VompA or VompC in E. coli could confer binding to
collagen IV. Both conferred the ability to bind collagen IV (Fig.
6A), but quantification revealed different collagen IV binding
efficiencies, with 6.6-fold greater binding by VompC than by
VompA (Fig. 6B; P ? 0.05). Thus, VompA and VompC are both
adhesins, and expression of either is sufficient to mediate binding
to the basement-membrane component of host cells, but with
different efficiencies.
Discussion
Virulence properties of Bartonella species include the ability to
persist in remarkably disparate hosts and niches and to adhere
to many different host-cell types (e.g., erythrocytes in the
mammalian bloodstream and epithelial cells in the arthropod
gastrointestinal tract). In the bloodstream of mammals, B.
quintana can cause prolonged, relapsing fever similar to Borrelia
species (5). Relapsing fever occurs when Borrelia surface pro-
teins undergo antigenic variation to circumvent the host immune
attack. To test our hypothesis that Bartonella persists by modi-
fying critical surface proteins during chronic infection, we first
developed an animal model to compare the repertoire of pro-
teins expressed in sequential bloodstream isolates. We identified
Fig. 3.
regions (PD, PAB, and PC) are shown in yellow. Based on the deduced amino acid sequences, highly conserved regions among the four genes are shown in blue,
MVregionsareshowninlightpurple,andminorvariable(mv)regionsareshowninlightorange.Theputativecollagen-bindingmotifs(NSVAIG-S)areindicated
by dark purple vertical lines within the MV regions of VompA, VompB, and VompC. Two tandemly repeated regions are shown in dark orange (x) and green (y).
Verticalarrowsindicatethepredictedleader-peptidasecleavagesites.(B)ThevomplocusinBQ2-D70hasonlytwogenes,vompDandvompC;vompAandvompB
have been deleted during the course of infection.
Schematic representation of the four vomp genes of JK-31 and the two genes of BQ2-D70. (A) Arrows indicate ORF and their orientations. Promoter
Fig. 4.
quintana isolates reveals heterogeneity in vomp gene copy number. Genomic
DNA from the B. quintana human (JK-31 and JK-5 to JK-63) and macaque
(BQ2-D70) isolates were digested with EcoRV and probed with a fragment of
theconserved5?regionofvompA,vompB,andvompC.Theprobebindstothe
two fragments of BQ2-D70 DNA flanking the vomp EcoRV site, indicating the
presence of vompC, and to four bands in JK-31, indicating the presence of
vompA,vompB,andvompC.InisolatesJK-5,JK-7,andJK-56,onlythreebands
are detected, suggesting the presence of only two copies of the conserved
region. In other human isolates, all three genes are present, but the region
flanking the vomp genes differs. There are a total of six different patterns
observed(I–VI,shownabovetherespectivelanes).Theprobecontrolisshown
in the far right lane.
Southern blot analysis of chromosomal DNA from nine human B.
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a family of four 100-kDa Vomp that are immunodominant and
variably expressed on the surface of B. quintana during pro-
longed bloodstream infection. Sequencing of the late isolate
BQ2-D70 revealed that vompA and vompB had been deleted.
Analysis of other B. quintana strains that were recovered from
humans revealed that vomp genes are deleted and?or rear-
ranged, implicating phase variation as a strategy used by B.
quintana to facilitate chronic human infection.
Although phase variation usually implies reversibility, it can be
irreversible, as reviewed by Henderson et al. (6). However, it is
critical that bacteria maintain the ability to express essential viru-
lence genes, such as adhesins. There are several mechanisms by
whichfunctionalvompgenescouldberestoredinthesettingofgene
deletion, including gene duplication or recombination either with
exogenously acquired B. quintana DNA or with vomp gene frag-
ments at other locations on the chromosomal DNA. The newly
released genome sequence for the B. quintana strain Toulouse
reveals vomp gene fragments downstream of vompC: one that is
tandemly arranged (similar to the fragment that we have identified
in JK-31) and one that is inverted (31). In addition, this strain has
only three complete vomp genes: vompD, vompC, and between
these two genes, a gene that is a mosaic of vompA, vompB, and
vompC, potentially representing some recombination event(s). All
of our isolates have at least two vomp genes, leaving the potential
for duplication or recombination between the remaining genes in
the locus or with fragments located at other sites on the chromo-
some (that were not detected by the probes used in our Southern
blot analyses). The mechanisms of deletion and recombination of
vomp genes require further investigation.
The Vomp have extensive N- and C-terminal amino acid and
secondary-structure homology with the Y. enterocolitica adhesin
YadA, including conservation of many of the YadA functional
domains. For YadA (20) and Vomp, the conserved C-terminal
domain is hydrophobic, has four transmembrane ?-strands, and
is predicted to serve as an anchor within the cell membrane (20).
Although VompD differs at the N terminus, it shares many
structuralfeatureswithVompA,VompB,andVompC,including
the conserved C-terminal membrane anchor. The extensively
conserved region just upstream of the predicted membrane
anchor domain of the Vomp is probably responsible for the
maintenance of a functional molecular structure, as with YadA
(20).ThetwoM.catarrhalisOMPadhesions,UspA1andUspA2,
have a secondary structural arrangement that is remarkably
similar to Vomp. UspA1 has head, neck, stalk, and membrane
anchor regions, as in Bartonella VompA, VompB and VompC;
UspA2 has the membrane anchor region but not the N-terminal
domains, similar to VompD (20).
Within the putative stalk domain of the C-terminal conserved
region of VompA, VompB, and VompC, there are two long,
tandemly arranged, repeated regions, in an X-Y—X-Y config-
uration. The function of these repeated regions is currently
unknown, but these regions could facilitate deletion of single or
multiple vomp genes (6). These tandem repeats also could be
involved in oligomerization of Vomp monomers, similar to the
YadA homooligomers resembling ‘‘lollipops’’ on the surface of
Yersinia (20). Analysis of the vomp locus in isolates from
additional humans and animals will provide insight into the
function of these regions in Bartonella.
Homology between Vomp and YadA led us to investigate the
adhesive properties of the B. quintana Vomp. YadA is the
prototype of a recently recognized class of proteobacterial
afimbrial adhesins that also includes the N. meningitidis adhesin,
NadA. In vivo, both function as virulence determinants essential
for establishing infection in animal models (22, 32). In vitro,
Fig.5.
to settle undisturbed for 36 h. JK-31 formed a pellet at the bottom of the tube (? tube). BQ2-D70 did not autoaggregate, and remained suspended (? tube). (B) For
quantitation,B.quintanastrainsweresuspendedinmedia,andtheOD600wasdetermined.TheOD600decreasedwithJK-31butnotBQ2-D70(P?0.05).(C)E.colistrains
were suspended in media, and the OD600was determined. Expression of VompA, but not VompC, in E. coli resulted in a significant decrease in the OD600compared
with the E. coli control (P ? 0.05). Data points represent the mean ? SEM of three independent assays done in duplicate (B. quintana) or triplicate (E. coli).
AutoaggregationprofileofJK-31,BQ2-D70,andE.colirecombinantstrainsexpressingVompAorVompC.(A)B.quintanastrainsweresuspendedandallowed
Fig. 6.
ability to bind collagen IV. (A) Coverslips were coated with collagen IV (Right)
or BSA control (Left). Expression of VompC in E. coli significantly increases
adhesion to collagen IV. Expression of VompA shows a moderate number of
E. coli adhering to collagen IV but significantly less than the E. coli expressing
VompC. (B) Quantitative analysis of the adherence of vomp recombinant E.
coli strains to collagen IV. Data represent the mean ? SEM of three indepen-
dentassaysdoneintriplicate.*,P?0.05,comparedwithE.colicontrol;**,P?
0.05, compared with both E. coli control and E. coli expressing VompA.
Heterologous expression of VompC or VompA in E. coli confers the
13634 ?
www.pnas.org?cgi?doi?10.1073?pnas.0405284101 Zhang et al.